| This study were designed to investigate development potency of the reconstructed embryos of black bear-cat inter-species nuclear transfer in vitro/vivo, to develop the principle and technology of somatic nuclear transplantation in inter-species of mammalian and to study the mechanism of coordination between cell nuclear and cytoplasm. Somatic nuclear transplantation technology has been used to establish stem cell from different animals, which can provide materials to treat various diseases. The main contents and results of the study are as follows: 1. The separation, culture and purification of black bear fibroblast cell for the donor of nuclear transferThe black bear fibroblast cell can be obtained from ear skin and neck skin of the black bear by issue culture method. The cells were cultured in DMEM and RPMI1640 mediums containing 10% fetal bovine serum(FBS), respectively. The two mediums can meet primary culture and passage culture of the black bear fibroblast cells. The method of single cell cloning by micro-manipulating purifiesfibroblast cells. As a result, steady fibroblast cells can be obtained and the rates of the cloning formation is 93.75%.2. Analysis of synchronization-treated bear fibroblast cells cycle by flow cytometryAfter 3 passage of the primary black bear fibroblast cells, the cells were serum starvated for 2-6 days or treated with 10g/ml Nocodazole for 24h, then calculation of percentages of the treated cells and the control cells in G0, G1, and G2+M phases of the cell cycle by using flow cytometry, respectively. GO+G1 cell percentage of the serum starvation group, Nocodazole treatment group and control group are 85.05+6.7,45.9+3.7 and 59.3+6.7, respectively. G2+M cell percentage are 10.4+6.8, 46.3+4.2 and 23.05+1.05, respectively. Black bear fibroblast cell treated with serum starvation for 3 days contained higher percentages of GO+G1, Whereas the cells treated with Nocodazole contained higher percentage of G2+M. The results of two synchronization treated groups and control group are notable (P < 0.05).3. Studies on superovulation in catsFifty nine cats (45 females, 5 males) were used in this study. Superovulation was induced by injection intramuscular of different dosages of FSH once daily for 5 days or FSH+30%PVP once, and then injection intravenous of 100 IU HCG on the 6th day. The cats were mated with ligated male cats at the time. The ovaries were examined and the oviducts and uteri were flushed in vitro by laparotomy for maturated oocytes recovery.The results were as follows: In comparison with control group, FSH group and FSH+30%PVP group are more efficient (p < 0.01), whereas the two treatment groups in equal dosage have no significant difference (p > 0.05). The optimal dosage of FSH for superovulation of the adult cats is 100 IU per cat, the recoverynumber of the maturated oocytes are 18.8+3.71, 19.1+2.77, respectively. 4. Somatic nuclear transfer of black bear-cat inter-species1) The donor of black bear fibroblast cells, which had been treated with serum starvation for 3d, 4d, 5d, and done with the 10g/ml Nocodazole for 24h, were used to recombine black bear-cat nuclear transfer embryos. After the recombined embryos were activated by 6-DMAP+CCB+ethanol, the development rates of morula were 20.22%, 18.31%, 17.65%, 9.76%, 6.89%(control) , respectively. The results show that the development rates of morula treated with serum starvation for 3d, 4d, 5d are higher apparently than Nocodazole group and control group (p < 0.05). whereas there are not difference in the development rates of morula of Nocodazole group and control group (p > 0.05).2) The study indicates that the enucleated rates of maturated oocytes in the medium containing 10g/ml CCB are better than the other dosages. the enucleated rates can reach at 87.06%.3) Three activated methods may effectively activate the black bear-cat reconstructed embryos. Whereas activated effect of ethanol+6-DMAP+CCB is more effective, the cleavage rate and the de...
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